Use of chlorogenic acid in preparation of drug for treating chordoma

Abstract

A drug that contains chlorogenic acid is effective in treating chordoma. When administered to a patient in need thereof, chlorogenic acid can significantly inhibit the proliferation of chordoma cells, reduce the expression level of multi-drug resistance gene MDR1 of chordoma cells, reverse the multi-drug resistance of chordoma cells, and effectively treat chordoma disease.

Claims

1. A method for treatment of chordoma, comprising administering an effective amount of a composition to a patient in need thereof, wherein the composition comprises chlorogenic acid at a dosage of 30-40 μmol/L.

2. The method according to claim 1, wherein preparation of said chlorogenic acid is an oral solution.

3. The method according to claim 1, wherein said chordoma is drug-resistant.

4. The method according to claim 3, wherein said drug-resistant chordoma is caused by MDR1 expressed by chordoma cells.

5. The method according to claim 4, wherein said chlorogenic acid is the one which can inhibit the proliferation of chordoma cells.

6. The method according to claim 2, wherein said preparation is a tablet.

7. The method according to claim 2, wherein said preparation is a freeze-dried powder injection.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 relative expression amount of MDR1 gene in human chordoma cells of different experimental groups.

EXAMPLES

Example 1 Preparation of Lyophilized Powder Injection with Chlorogenic Acid

(2) 1. Extraction of Chlorogenic Acid

(3) The raw material of chlorogenic acid used in this example is extracted and purified from leaves of Eucommia ulmoides, with a purity of 99.6%.

(4) 2. Preparation of Lyophilized Powder Injection of Chlorogenic Acid

(5) 2.1 Formula

(6) TABLE-US-00001 Chlorogenic acid (principal agent) with a purity of 99.6% 30 g Mannitol (support agent) 50 g Vitamin C (antioxidant) 10 g

(7) 2.2 Preparative Method

(8) Above formula was thoroughly dissolved in the water for injection. After filtration, 0.22 μm microfiltration membrane for removing bacteria was further used for fine filtration. After adjusting pH, 1000 of 2 ml powder injections were prepared according to the common procedure of sterile lyophilized powder. Each injection contained 30 mg chlorogenic acid, of which the content of chlorogenic acid was 33.3%.

Example 2 Preparation of Oral Solution with Chlorogenic Acid

(9) 1. Extraction of Chlorogenic Acid

(10) Chlorogenic acid used in this example is extracted and purified from Flos lonicerae, with a purity of 98.47%.

(11) 2. Preparation of Oral Solution with Chlorogenic Acid

(12) 2.1 Formula

(13) TABLE-US-00002 Chlorogenic acid (principal agent) with a purity of 98.47% 22.5 g Vitamin C (antioxidant) 15 g Water for injection (solvent) 10 L

(14) 2.2 Preparative Method:

(15) The amount of chlorogenic acid indicated in the formula and Vitamin C were taken out and dissolved in 10 L water for injection. According to the conventional preparative process of oral solution, after filtration, the solution was aseptically filled to obtain 100 bottles of oral solution, and each bottle contained 100 mL solution, i.e. 225 mg chlorogenic acid, of which the content of chlorogenic acid is 60%.

Example 3 Preparation of Tablets with Chlorogenic Acid

(16) 1. Extraction of Chlorogenic Acid

(17) Chlorogenic acid used in this example is extracted and purified from Flos lonicerae, with a purity of 99.3%.

(18) 2. Preparation of Tablets with Chlorogenic Acid

(19) 2.1 Formula

(20) TABLE-US-00003 Chlorogenic acid (principal agent) with a purity of 99.3% 100 g Sugar powder (bulking agent) 100 g Lactose (bulking agent) 200 g Hydroxypropylmethylcellulose (binding agent)  50 g Magnesium stearate  50 g

(21) 2.2 Preparative Method:

(22) In this example, chlorogenic acid tablet is prepared by wet granule pressing method. (1) The amount of hydroxypropylmethylcellulose as indicated in formula was taken out and dissolved in water to prepare water solution; (2) Chlorogenic acid, sugar powder and lactose were taken out as the amount shown in formula, mixed, and added to hydroxypropylmethylcellulose aqueous solution, then the mixture was well mixed to prepare soft material; (3) According to the conventional operation procedure of wet granulation, the resultant soft material was sieved, dried, and pelletized to get uniform size particles; (4) The resultant particles were mixed with magnesium stearate and pressed, thereby 1000 tablets were prepared, and each tablet contained 100 mg chlorogenic acid, in which the content of chlorogenic acid is 20%.

(23) In the following, the beneficial effect of the present invention was proved by specific pharmacodynamic experiments:

Experimental Example 1 the Inhibition of Chlorogenic Acid on the Proliferation of Human Chordoma CM-319 Cells

(24) 1. Materials

(25) Drug: chlorogenic acid raw material (99.6% purity, Sichuan Jiuzhang Biotechnology Co., Ltd.) Cell line: human chordoma cell line CM-319 cells (West China Hospital, Sichuan University)

(26) 2. Experimental Method

(27) Human chordoma CM-319 cells growing in logarithmic phase was collected, and the cell concentration was adjusted to 6×10.sup.5 cells/ml with 1640 medium. 100 μL suspension of above cells was taken out and inoculated into 96 well plate, and each experimental group has three replicate wells. The detailed groups are shown in Table 1. To each well, was added 100 μL corresponding drugs, so that the final concentration of chlorogenic acid in the experimental group is 10 μmol/L, 20 μmol/L, and 40 μmol/L, respectively, and then the plate was further cultured for 24 hours. In the negative control group, no drug was added, and only the same volume of 100 μL culture medium was added. After 24 hours, 10 μL CCK-8 solution was added to each well, and the culture plate was incubated in the incubator for another one hour. The absorbance of each well was measured at 450 nm by ELISA. The inhibition of chlorogenic acid on human chordoma CM-319 cells was calculated at various concentrations by using the growth inhibition rate=(absorbance of the negative control group at 450 nm—absorbance of the experimental group at 450 nm)/absorbance of the negative control group at 450 nm×100%.

(28) TABLE-US-00004 TABLE 1 Different experimental groups and chlorogenic acid concentrations. Experimental Concentration of groups chlorogenic acid (μmol/L) Group 1 10 Group 2 20 Group 3 40 Group 4 0 (negative control group)

(29) 3. Experimental Results

(30) The proliferation of human chordoma CM-319 cells treated with different concentrations of chlorogenic acid was calculated, and the results are shown in Table 2:

(31) TABLE-US-00005 TABLE 2 The inhibitory effect of chlorogenic acid at different concentrations on the proliferation of human chordoma CM-319 cells Experimental Inhibitory rate groups of cells (%) Group 1 36.77 ± 5.12 Group 2 48.37 ± 6.11 Group 3 55.49 ± 3.78

(32) In this example, CCK-8 test kit was used to detect the effect of chlorogenic acid on the proliferation of human chordoma CM-319 cells. The experimental results show that different concentrations of chlorogenic acid can strongly inhibit human chordoma cells and prevent the proliferation of cells, and this inhibition can improve as the increase of chlorogenic acid concentration, suggesting that chlorogenic acid can inhibit the growth of human chordoma cells, and this inhibition is dose-dependent to some extent.

Experimental Example 2. Effect of Chlorogenic Acid on MDR1 Expression in Human Chordoma Cells CM-319

(33) 1. Materials

(34) MDR1 primer (Shanghai Bioengineering Co., Ltd.); RNA extraction kit (Jiangsu biyuntian); reverse transcription synthesis kit (Jiangsu biyuntian); chlorogenic acid API (purity 99.6%, Sichuan Jiuzhang Biotechnology Co., Ltd.).

(35) 2. Experimental Method

(36) In this example, RT-PCR was used to detect the effect of different experimental groups on the expression of MDR1 in human chordoma CM-319 cells. The detailed experimental groups are shown in Table 3. CM-319 cells cultured in 6-well plate was collected, and RNA extraction kit was used to extract total RNA in cells, then reverse transcription synthesis kit was used to transcribe the first strand of cDNA. After that, PCR amplification reaction was used to detect the expression level of target genes in different experimental groups.

(37) TABLE-US-00006 TABLE 3 Different experimental groups and chlorogenic acid concentrations. Experimental Concentration of groups chlorogenic acid (μmol/L) Group 1 10 Group 2 20 Group 3 40 Group 4 0 (Negative control group)

(38) 3. Experimental Results

(39) The results of RT-PCR showed that different concentrations of chlorogenic acid could reduce the expression level of MDR1 in CM-319 cells to various degrees, in which high concentration of chlorogenic acid (40 μmol/L) could significantly reduce the expression level of drug-resistant genes in cells compared with other groups, and further improve the sensitivity of tumor cells to drugs. The test results are shown in FIG. 1.

(40) In summary, chlorogenic acid can significantly inhibit the proliferation of chordoma cells, reduce the expression level of MDR1 gene in chordoma cells, reverse the multidrug resistance of chordoma cells, effectively treat chordoma, and has a good clinical application prospect.